Preparation of polyphenol-rich dried biomass extracts

Abstract

The present invention relates to a method for preparing a polyphenol-rich extract of dried plant biomass, in particular of parts of grapes, the extract obtained and its use for antifungal applications, in particular for the prevention and treatment of fungal infections of post-harvest fruits and plants, and in connection with its antibacterial and antioxidant properties.

Description

[Technical field]

[0001] The present invention relates to a process for the preparation of a polyphenol-rich extract of dried plant biomass, in particular of parts of grapes, the extract obtained and its use for antifungal applications, in particular for the prevention and treatment of fungal infections of post-harvest fruits and vegetables, and in connection with its antibacterial and antioxidant properties. [Background technology]

[0002] Polyphenol-rich extracts of plant biomass are known to those skilled in the art. Many studies have focused on extracting biomass to obtain polyphenol-rich extracts, but they all have some drawbacks in terms of the economic viability of the methods, such as low yields or very long extraction times, their limited use after extraction, especially the use of toxic organic solvents (Goupil et al., 2012; Alexa et al., 2012; Sanchez et al., 2008; Pezet et al., 2004).

[0003] Various polyphenol extraction techniques and uses are described, inter alia, in patents and patent applications US2012 / 0021080, US5,989,557, US2012 / 0142105, JP2016 / 102192, CN205 759 861, CN103 875 842, or in articles by Osorio et al. (2010), Luque-Rodriguez et al. (2006), or Favaron and Lucchetta (2009).

[0004] Studies have shown that it is possible to improve biomass extraction methods by microwave, ultrasound or high temperature treatment (Quan et al., 2006, CN104 177 463, CN104 435 135, CN102 757 512, CN104 256 432, CN104 256 641, CN103 783 506, CN102 757 509, CN101 816 349), for example for the production of biodiesel (CN19 35 947). Some recommend high temperature treatment under high pressure (Casazza et al., 2010).

[0005] However, such high temperature treatments may alter the properties of the resulting extract and are not suitable for implementing industrial processes for large amounts of raw materials and solvents.

[0006] There is a need for an efficient method for biomass extraction to prepare polyphenol-rich extracts that is efficient, more economical, and does not affect the properties of the biomass with respect to its polyphenols. Summary of the Invention [Means for solving the problem]

[0007] The present invention relates to a method for preparing an extract of a dried plant biomass, in particular a polyphenol-rich extract of grape species, comprising the steps of (a) extracting the dried biomass by contacting the dried biomass with an aqueous solvent and (b) recovering a polyphenol-rich aqueous phase, wherein in the extraction step (a), the plant biomass / aqueous solvent mixture comprises: (i) Electromagnetic waves with frequencies in the range of 915 MHz to 28 GHz; (ii) stirring the mixture; and / or (iii) Pressure between 50 and 950 mbar (5,000 and 95,000 Pa) The present invention relates to a method for treating a subject with a combination of the above-mentioned compounds simultaneously or successively.

[0008] The present invention also relates to the extract obtainable by this method and its use for antifungal applications, in particular for the prevention and treatment of fungal infections of post-harvest fruits and plants. [Brief description of the drawings]

[0009] [Figure 1]Figure 1 shows a comparison of the relative amounts of trans-piceatannol, trans-resveratrol, trans-ε-viniferin and trans-vitisin contained in extracts obtained by the method according to the invention and by conventional methods, where the values ​​shown represent the relative concentrations (arbitrary units) of each of the four molecules, calculated from the areas under the curve of the HPLC peaks. [Diagram 2] Figure 1 shows a comparison of the antifungal activity of extracts obtained by the method according to the invention (circles) and by conventional methods (squares). The values ​​shown are the average of the efficacy in three identical conditions (0% = normal fungal growth; 100% = no fungal growth) at day 4 after inoculation as a function of the extract concentration. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The present invention relates to the preparation of a polyphenol-rich extract by extracting dried biomass with an aqueous solvent.

[0011] According to the present invention, the term "aqueous solvent" means water or an aliphatic alcohol / water mixture, in particular ethanol / water. In this case, the alcohol / water mixture, in particular ethanol / water, contains 10-70% by volume of ethanol. According to a preferred embodiment of the present invention, the aqueous solvent is an ethanol / water mixture containing 15-40% ethanol.

[0012] The plant biomass according to the invention is a biomass known to contain polyphenols, in particular trans-resveratrol, trans-ε-viniferin, trans-piceatannol and trans-vitisin, per se. The skilled artisan is familiar with this biomass from plants known to contain these polyphenols, generally dicotyledons such as grape, tea and Japanese knotweed, in particular grape, used alone or in mixture. For the biomass according to the invention, the whole plant or parts of the plant are used, more particularly the above-ground parts of the plant, such as fruits, flowers, leaves, stems, branches or pieces of the trunk (vines), and mixtures thereof. If necessary, the biomass may also include or consist of the roots of the plant.

[0013] The biomass is dried, i.e. obtained by drying the plants or plant parts as described above. Drying can be carried out by any method known to the person skilled in the art. Advantageously, drying is achieved by drying the plants or their parts in the open air. According to a preferred embodiment of the invention, the plants or their individual parts are dried in the open air for a period between 1 month and 4 years, preferably between 1 and 2 years.

[0014] Preferentially, the dry biomass consists of dry parts of plants of Vitis vinifera, Vitis labrusca, Vitis riparia, Vitis rupestris, Vitis berlandieri, Vitis amurensis, Vitis coignetiae, Vitis vulpina, Vitis acerifolia, Vitis aestivalis and Vitis rotundifolia, in particular Vitis vinifera plants. According to an advantageous embodiment of the invention, the dry biomass comprises or consists of grape shoots.

[0015] Advantageously, this dry biomass is composed of plant fragments, in particular comminuted plants or plant parts. Preferentially, the fragments have a particle size of less than 1 cm, more preferentially less than 5 mm, and even more preferentially in the range of 1-5 mm. The particle size of this dry matter fragment / comminuted dry matter is measured according to the usual methods known to the skilled person.

[0016] Fragmentation of the dried plant material is carried out according to the general methods known to those skilled in the art. According to a particular embodiment of the invention, these fragments are obtained by first crushing the dried material to obtain fragments of 1-20 cm, which are then crushed to obtain fragments of preferably 1-5 mm, corresponding to the particle size preferred for extraction.

[0017] The skilled person is able to determine the amount of extraction solvent to be used relative to the amount of biomass. Advantageously, the mass ratio of dry plant biomass to aqueous solvent ranges from 1 / 5 to 1 / 30, preferentially from 1 / 10 to 1 / 20.

[0018] The method according to the invention is characterized by the combination of at least two stresses applied simultaneously or successively to the extraction mixture (biomass / solvent): (i) Electromagnetic waves with frequencies in the range of 915 MHz to 28 GHz; (ii) stirring, and / or (iii) Pressure between 50 and 950 mbar.

[0019] According to the invention, the expression "simultaneous or successive stirring" means that the microwave treatment, stirring and / or pressure at least below 950 mbar are carried out simultaneously or one after the other, in particular according to the sequence (i)+(ii), or (i)+(iii), or (i)+(ii)+(iii), or [(i),(ii)]+(iii), or [(ii),(iii)]+(i) in any order. In the case of successive treatments, the skilled person can choose to repeat each sequence several times or to combine different sequences according to a given program, possibly in combination with one or more simultaneous treatment sequences.

[0020] According to a preferred embodiment of the present invention, the three processes are carried out simultaneously [(i), (ii), (iii)].

[0021] Step a) may further comprise an ultrasonic treatment (iv), which may be applied sequentially or simultaneously with the treatments (i), (ii) and / or (iii) defined above.

[0022] Electromagnetic waves with a frequency in the range of 915 MHz to 28 GHz correspond to waves in the microwave range. A person skilled in the art can select the most suitable frequency for carrying out the method, in particular depending on the biomass, in order to optimize the extraction method. The frequency of the electromagnetic waves is 915 MHz to 2.45 GHz. Advantageously, the frequency of the electromagnetic waves is selected according to known industrial devices for generating electromagnetic waves on an industrial scale, in particular generators with a frequency of less than 1500 MHz, more advantageously about 915 MHz.

[0023] The skilled person can further select the wave power that is most suitable for the biomass and the extract to be obtained. The power of the electromagnetic waves is in the range of 300 W to 100 kW, more preferably 1 to 75 kW.

[0024] This power is selected in particular according to the amount to be processed and the time required for extraction. According to a preferred embodiment of the invention, the power of the electromagnetic waves is selected according to a ratio of 1 to 10 kW, ideally 2 to 6 kW, per kg of material to be extracted.

[0025] Advantageously, the extraction time is at least 20 minutes, or even at least 30 minutes, preferably less than 125 minutes, understanding that too short a time may be associated with overpowering conditions that may alter the biomass, while too long a time may result in deterioration of the extraction product, in either case with a risk of polyphenol loss.

[0026] The method according to the invention is suitable for use with different amounts of biomass / solvent mixture, in particular at least 1 L, preferentially at least 5 L. The method according to the invention is particularly suitable for industrial extraction of dry biomass. Advantageously, the method is used with a biomass / solvent mixture volume of at least 30 L, which may range from more than 50 L, or even more than 100 L. Depending on the extraction reactor available to the person skilled in the art, the method according to the invention can be used with volumes of 200 L or more.

[0027] The biomass / solvent mixture is subjected to stirring (ii), which is preferentially carried out simultaneously with the electromagnetic wave treatment (i), which stirring is important to allow the electromagnetic waves to reach the entire mixture, otherwise only the surface of the mixture would be subjected to the action of these electromagnetic waves.

[0028] The skilled person knows suitable means for carrying out the stirring operation according to the amount of mixture to be treated. In particular, means for rotating the mixture to be treated, generally by means of a rotating blade within the mixture or by rotating a reactor containing the mixture to be treated, are known. The skilled person may also consider other stirring means, such as moving the mixture by bubbling air or an inert gas. Preferentially, the stirring is carried out by rotating the mixture, more preferentially by a rotating blade within the mixture, at a rotation speed that may range from 3 to 20 rpm.

[0029] Those skilled in the art can select the most suitable rotation means for operation in an environment susceptible to electric arcing and subject to electromagnetic wave treatment.

[0030] The method also preferably comprises subjecting the mixture to a vacuum (iii) during the microwave treatment (i) and stirring (ii).

[0031] The skilled person is familiar with the means necessary to obtain a vacuum (or partial vacuum) in a closed vessel containing the mixture to be treated. These industrial means make it possible to obtain pressures in the range of 50 to 950 mbar (5,000 to 95,000 Pa). The skilled person can select the appropriate pressure depending on the quantities to be treated. In particular, for the industrial-scale quantities mentioned above, it is advantageous to treat the mixture at a pressure of 50 to 950 mbar (5,000 to 95,000 Pa), preferentially 50 to 500 mbar (5,000 to 50,000 Pa).

[0032] Since the method also involves ultrasonic treatment, the ultrasonic frequency and power can be selected by the skilled person. Advantageously, the ultrasonic frequency is in the range of 25 kHz to 1 MHz. According to a preferred embodiment of the invention, the ultrasonic power is in the range of 200 to 4000 W.

[0033] The present invention further relates to the process as defined above and in the examples, comprising an additional step (c) of concentrating the polyphenols by partial or total evaporation of the aqueous solvent.

[0034] According to a preferred embodiment of the invention, this concentration (c) is carried out by freeze-drying the recovered aqueous phase.

[0035] According to a particular embodiment of the invention, the dried biomass is subjected to leaching with a suitable solvent to remove sugars prior to the extraction step (a). The skilled person can determine the conditions of this leaching depending on the dried biomass and its sugar content.

[0036] The removal of sugars depending on the biomass being treated is important to obtain extracts rich in polyphenols with antifungal properties. Indeed, it has been shown that the presence of sugars can have an antagonistic effect on the antifungal activity of polyphenols by promoting fungal growth.

[0037] The invention further relates to polyphenol-rich biomass extracts obtained by the method according to the invention. These extracts advantageously have the four molecules of interest, which are polyphenols of the stilbene series: trans-piceatannol, trans-resveratrol, trans-ε-viniferin and trans-vitisin. Preferably, the extract contains at least 2 times more (or at least 2.5 times more) trans-ε-viniferin and at least 3 times more (or at least 6 times more) trans-vitisin than conventional ethanol extracts.

[0038] The invention further relates to a polyphenol-rich biomass extract obtainable by the method according to the invention for a biomass consisting of the dry parts of plants of Vitis species, in particular Vitis vinifera, Vitis americana, Riparia spp., Rupestris spp., Berlandieri spp., Vitis manchuria, Vitis vulpina spp., Acerifolia spp., Aestivalis spp., Muscadine spp., in particular Vitis vinifera. According to a preferred embodiment of the invention, the polyphenol-rich extract is an extract of grape shoots, more particularly Vitis vinifera shoots.

[0039] The extracts according to the invention have fungicidal, bactericidal and antioxidant properties, as shown by the example of an extract of Vitis violaceum shoots.

[0040] The extracts according to the invention, in particular the grape shoot extracts, are effective as fungicides for controlling (preventing) the occurrence of harmful fungi in food and agriculture, such as contaminants in canned fruit (Byssochlamys nivea) or diseases of fruit trees, vegetables and plants, for example apple canker (Nectria galligena), brown rot of fruit trees (Monilinia fructicola, Monilinia fructigena and Monillinia laxa), carrot blight (Alternaria daucil), potato silver scab (Helminthosporium solani), Fusarium head blight (Gibberella zeae, Fusarium culmorum), Septoria leaf blight of wheat (Mycosphaerella graminicola) or various rot diseases (including Penicillium digitatum, Penicillium italicum, Phytophthora syringae or Rhizopus stolonifer).

[0041] The present invention further relates to a method for the fungicidal treatment of plants in order to prevent or inhibit the occurrence of pathogenic fungi in said plants, said method comprising applying to said plants an effective amount of an extract according to the invention, in particular a grape shoot extract as defined above and in the examples.

[0042] Plants which can be treated with the extract according to the invention are advantageously plants cultivated for human consumption, such as market gardens, potato crops, agricultural crops, such as cereals, in particular barley, wheat and corn, soybean or oilseed crops, such as rapeseed, sunflower, flax, soybean or cotton. The treatment can also be applied to fruit trees, in particular apple, pear, apricot, peach and vines.

[0043] Application to plants is carried out as required, either as a preventive measure when climatic conditions are favorable for the development of fungi, or as a therapeutic measure after the appearance of disease. Application is generally carried out using standard spraying methods. Those skilled in the art can determine when and how to treat plants with the extract according to the invention, used alone or mixed with other products known for their fungicidal properties, such as copper sulfate, Bordeaux mixture, or other plant protection products from the agrochemical industry. Advantageously, treatment is carried out without the use of plant protection products from the agrochemical industry, for organic or biodynamic agriculture.

[0044] The present invention further relates to a method for preventing and / or treating post-harvest deterioration, in particular the occurrence of fungal decay, of fruits and plants, which method comprises applying to said fruits and plants an appropriate amount of an extract obtainable by the method according to the invention as defined above and in the examples.

[0045] Due to their antioxidant properties, the extracts according to the invention, in particular the grape shoot extracts, can also be used to preserve food compositions or cosmetic compositions.

[0046] Due to its ability to slow down the oxidation of fruits after harvest, the extract according to the invention, in particular the grape extract, can be used to prevent and / or treat oxidative stress in plants by application to these plants or parts of plants, in particular to oxidation-sensitive fruits such as bananas, which are prone to oxidative stress when stored at lower temperatures than those found in their natural environment, or to exotic or tropical fruits such as mango, passion fruit, etc. The invention further relates to the use of the extract according to the invention, in particular the grape shoot extract, or a composition containing it, for the prevention and / or treatment of oxidative stress in plants.

[0047] The extract according to the invention, in particular the grape shoot extract, can be used due to its antioxidant properties for the prevention and treatment of oxidative stress in humans and animals.

[0048] They also have antibacterial activity, especially against pathogenic Gram-positive bacteria, while preserving the commensal bacteria in humans. They are particularly effective against bacteria of the genera Staphylococcus, Listeria, Streptococcus and Propionibacterium, especially Propionibacterium acnes, the pathogen responsible for acne. The extracts according to the invention, especially the grape shoot extracts, have a broad spectrum of antibacterial activity and are more effective against skin pathogens.

[0049] The present invention therefore relates to an extract according to the invention, in particular a grape shoot extract, for use in therapy, in particular for the treatment and prevention of bacterial infections, more particularly for the treatment and prevention of bacterial skin infections, and even more particularly for the treatment and prevention of acne.

[0050] The present invention further relates to a composition comprising an extract obtainable by the method according to the invention as defined above and in the examples, and a carrier suitable for use.

[0051] This may be an antifungal or pharmaceutical composition for application to plants pre- or post-harvest. Such compositions are known and the skilled artisan can determine suitable carriers for this use.

[0052] The present invention particularly relates to a plant health composition comprising at least the above extract and at least one compatible compounding agent, which may be in dry form, such as a powder or granular form, or in liquid form, such as a suspension, concentrated or non-concentrated dispersion, gel, emulsion, etc.

[0053] The compounding agents are derived from natural agents or synthetic chemistry and are commonly used in plant health compositions.

[0054] The compounding agents may be dispersants, stabilizers, surfactants, preservatives, wetting agents, adhesives, buffers, pH adjusters, light protectants, etc. These may be used alone or in admixture.

[0055] The extracts obtainable by the method according to the invention, as well as compositions containing them, are particularly suitable for use in the prevention and treatment of fungal infections of post-harvest fruits and plants.

[0056] The method according to the invention involves the implementation of several combined techniques in the same extraction medium. Each technique (electromagnetic, ultrasonic, vacuum) and the means for implementing them are well known (WO 2012 / 045923, EP 2 530 059, FR 2 976 062). Those skilled in the art can adapt existing hydroalcoholic extraction systems for dry biomass to apply these related processes. EXAMPLES

[0057] Example 1 Preparation of Vitis vinifera shoot eco-extract: The starting plant material is Vitis vitis shoots (August shoots). The shoots are dried (in the open air or in an oven). Once dry, the shoots are first chopped into pieces of 2-10 cm and then finely ground to a particle size ranging from 1-5 mm. The Vitis vitis shoot powder thus obtained is extracted in a 30% aqueous or ethanolic solution. The extraction time ranges from 30 minutes to 1 hour and 30 minutes, preferentially from 45 minutes to 60 minutes. The techniques used for the extraction are microwave, ultrasonic, vacuum and simultaneous stirring. The extract thus obtained is vacuum filtered through a 20 micron filter. The extract is then evaporated under vacuum and spray-dried or freeze-dried. The dry extract thus obtained is then stored at room temperature protected from light.

[0058] Example 2 Chemical characterization of the Vitis vinifera shoot extract prepared according to Example 1 5 mg of freeze-dried dried Vitis vinifera shoot eco-extract obtained according to Example 1 was dissolved in 1 ml of 50% ethanol. After solubilization, the solution is centrifuged at 12000 RCF for 10 min and loaded into a high performance liquid chromatography / mass spectrometry (HPLC-MS) system under the following conditions: the amount of extract loaded is 25 μl; the mobile solvents are ultrapure water (0.1% formic acid) and acetonitrile (0.1% formic acid). The separation is performed at 1 ml / min for 55 min according to the following solvent gradient:

[0059] [Table 1]

[0060] The column used contains a stationary phase grafted with C18 functional groups. UV detection is performed between 200 and 800 nm. Mass detection is performed by negative mode ESI. Four main compounds are obtained, their retention times, absorbance and molecular weights are shown in the table below.

[0061] [Table 2]

[0062] A comparison of the contents of these four compounds with that of a conventional ethanol extract is shown in Figure 1.

[0063] Example 3 Antifungal effect against Botrytis cinerea: Botrytis cinerea spores are allowed to settle to the bottom of the wells of a clear 96-well plate that has been pre-filled with agar nutrient medium.

[0064] The freeze-dried dry Vitis vinifera shoot eco-extract obtained according to example 1, dissolved in 8% ethanol at different concentrations (0 g / l, 2.5 g / l, 5 g / l, 10 g / l, 20 g / l and 30 g / l), is then deposited in the wells.

[0065] After various incubation times at 21° C., protected from light, the relative density of the mycelium in each well is measured by absorbance at 800 nm. The antifungal effect is then calculated from these absorbance values.

[0066] The antifungal effect observed for extracts obtained by the method according to the invention and by the conventional method after 4 days of incubation is shown in Figure 2. The results are at least similar to those shown in Figure 2 for up to 15 days of incubation.

[0067] Example 4 Antifungal effect The minimum inhibitory concentration (MIC) with 100% efficacy up to 240 hours was determined against different fungal strains using the extract according to Example 2. The results are detailed in the table below.

[0068] [Table 3]

[0069] Example 5 Antibacterial effect The MICs of the extract of Example 2 were determined against different bacterial species. The tests are carried out against 27 strains, as listed in the table below. The tests are carried out at a concentration of 5·10 5 ~10 6 This is done in high nutrient medium calibrated to CFU / mL. [Table 4] TIFF0007645605000005.tif243164

[0070] The extract of Example 2 shows inhibitory activity against all tested strains except for P. plantarum and Vibrio anguillarum. The most sensitive strain tested is P. acnes, the pathogen causing acne. Finally, fungi are most sensitive to the extract of Example 2 in the order of Bacillales, Enterococcus species, and certain Streptococcus species. On the other hand, the other tested strains, especially the beneficial Bifidobacterium species and Lactobacillus species, are less sensitive, as are the Gram-negative bacteria.

[0071] The extract of Example 2 is a broad spectrum antibacterial agent and has higher efficacy against skin pathogens.

[0072] Example 6 Antioxidant Activity The antioxidant activity of the extract of Example 2 was examined by comparing the condition of banana peels placed outdoors with or without the extract of Example 2 applied on the 7th and 9th days after application.

[0073] When the extract of Example 2 was not applied, the banana skins were observed on the 7th day to be covered with brown spots characteristic of their oxidation, whereas the treated banana skins were only slightly affected (one characteristic spot).

[0074] By day 9, oxidation of the untreated banana peels had continued from the speckled state observed on day 7 to a completely blackened state, while the treated banana peels remained largely unchanged from the state observed on day 7.

[0075] This antioxidant activity observed in bananas indicates the benefit of the extract according to the invention for treating oxidative stress in plants, especially in exotic or tropical fruits.

[0076] [Table 5] -

Claims

1. A method for preparing a polyphenol-rich extract from lignified branches, which are the dry parts of grapes, comprising the steps of: (a) extracting a component containing polyphenols from the lignified branches, which are the dried parts of the grapes, by contacting the lignified branches, which are the dried parts of the grapes, with an aqueous solvent; (b) recovering the polyphenol-rich aqueous phase; (c) concentrating the polyphenols by partially or totally evaporating the aqueous solvent, The crushed lignified branches, which are the dry parts of the grapes, have a particle size in the range of 1 mm to 1 cm; In the extraction step (a), the lignified branches / aqueous solvent mixture, which is the dried part of the grape, (i) electromagnetic waves with a frequency in the range of 915 MHz to 2.45 GHz and a power of 1 to 10 kW per kg of material to be extracted; (ii) stirring the mixture with a rotating blade at a rotation speed in the range of 3 to 20 rpm; are simultaneously processed by the aqueous solvent is an ethanol / water mixture containing 10 to 70% by volume of ethanol, and the mass ratio of the lignified branch, which is the dried part of the grape, to the aqueous medium is 1 / 5 to 1 / 30; The extraction time in step (a) is 20 minutes to 125 minutes; The method according to claim 1, wherein the polyphenols include resveratrol, viniferin, and vitisin.

2. The method of claim 1, wherein the aqueous solvent is an ethanol / water mixture containing 30-50% ethanol.

3. The method according to claim 1 or 2, wherein the mass ratio of the lignified branches, which are the dry parts of the grapes, to the aqueous solvent is 1 / 10 to 1 / 20.

4. The method according to any one of claims 1 to 3, wherein the ground lignified branches, which are the dry parts of grapes, have a particle size in the range of 1 mm to 5 mm.

5. The method according to any one of claims 1 to 4, wherein the output of the electromagnetic waves is in the range of 300W to 100kW.

6. The method according to claim 5, wherein the electromagnetic wave power is in the range of 1 to 75 kW.

7. In the extraction step (a), the lignified branches / aqueous solvent mixture, which is the dried part of the grape, (i) electromagnetic waves with frequencies in the range of 915 MHz to 2.45 GHz; (ii) stirring the mixture; and (iii) a pressure of 5,000 to 50,000 Pa (50 to 500 mbar); 7. The method according to claim 1, wherein the first and second substrates are simultaneously treated with

8. The method of any one of claims 1 to 7, wherein said concentrating (c) is accomplished by freeze-drying the recovered aqueous phase.

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